Cell biology Nitric oxide early stress signals and cell death

Improvements in instrumentation are needed to observe and interpret how UV-radiation affects the cellular networks of plants. Modeling of the dose-responses should take advantage of the insights from functional plant genomics [2] and from new virtual imaging technologies [3]. Most current genomic information is based on a flowering mustard plant and on rice. By 2010 the function of every gene in the Arabidopsis plant will be worked out. Will we be able to apply this information to other agricultural plants and to forest trees? How can we benefit from data that shows the function of every gene? Where and how are UV-sensitive genes regulated and expressed? How do they interact with each other to give us useful information? How can this information be used to advance our understanding of phenetics or the understanding of trait, population and evolution [4], and environmental health?

Some microscopes designed for in vivo imaging now employ two-photon fluorescence and second-harmonics. This enables the imaging of cellular membranes and their action potentials in live cells. Two-photon tracking reveals the phased transport of molecules in images 250 p.m wide. Microscopes can also image to a depth of ca 50 p.m, a spatial resolution of 0.6 p.m, and a temporal resolution of 0.833 ms. The measurements of the behavior of single molecules now require small sample volumes. However, these assays are costly to perform [5, 6].

The free radical, nitric oxide (NO) is a very early and highly transient metabolic signal in the stress response of cells. Laser confocal microscopy and detecting dyes, or laser photoacoutsic detection, have been effective for monitoring NO bursts in cells and plants respectively. NO is lipid soluble and diffuses in membranes. It leads to protective and or harmful reaction depending on the perturbing conditions. Damaging effects arise from the reaction of NO with other free radicals that may not be produced by enzymatic reactions [7]. With optical methods, the problems due to photodamage and toxicity must be evaluated and ruled out. Fluorescent sensors and combined techniques have produced consistent models to quantify stress and damage [8].

NO is derived from nitrate in the cell, or from an enzyme that uses L-arginine and oxygen to produce NO and citrulline. Historically, the organization of reactions of into amino acid families has employed state-network maps to assess the responses of fruit and forest trees to environmental, nutritional, and pathological changes under field conditions [9,10]. The mapping of physiological states over time will now benefit from the emerging models in metabolomics. Flux-balance analyses have shown that metabolic networks are dominated by several reactions with very high fluxes [11]. Results with Escherichia coli are believed by some to represent a universal feature of metabolic activity of all cells. Improved and more specific probes are needed to determine the 'time of flux' for NO reactions leading to UV protection and damage.

In plant development, cell death (apoptosis) performs a myriad of necessary functions from sculpting out organ shapes to adapting plants to various stresses throughout their life histories [12]. Understanding the developmentally programmed cell-death signals and their manifestation is a key step in designing monitoring systems not only for UV radiation, but also for the electromagnetic spectrum.

In the human body, damage due to UV-B radiation may involve the p53 tumor-suppressor protein. This represents one of the most effective natural defenses against cancer. Small-molecule drugs have been designed to activate p53 by preventing the binding of negative regulators of p53 for a new and effective genotoxic general treatment for cancer [13]. While plants are considered to lack of p53 homolog, proteins having partial amino acid sequences related to human p53 and to the mitotic inhibitor p21 have been detected in conifer cells cf. Durzan, Santerre, and Havel; Rotari et al. (these proceedings). We do not know if proteins and other metabolites produced by plants under UV-B radiation would offer new pharmacogenomic insights in the treatment of malignancies.

The set of external signals received by a plant cell creates a brief instability that usually consumes a mere fraction of the energy expended relative to the final outcome. After the response to a stimulus, the original physiological state is assumed to return and ready to again respond. However, prior responses may change the ability of a cell or site to respond later. This fatigue (time needed for recovery) may require a summation of prior stimuli. Hysteresis in the response curves becomes evident by the retardation of repeated responses and can lead to the aging of cells.

The time elapsed between the beginning of a stimulus and the end reaction is call the 'reaction time'. It is made up of the perception time (e.g., a small fraction of a second in a cell), the transmission time (e.g., 0 to 4 cm per second in a plant), and the physiological response, which is variable and far longer depending on the systems response as in the case of morphogenesis. Internal plant correlations that synchronize and coordinate the physiological rhythms and display a 24-hour periodicity set by light-dark cycles.

The 'metabolic pursuit' or 'tracking curves' of nonlinear fluctuations have a long history in hereditary mechanics [14]. However, when the number (n) of external variable exceeds 7, the phenotypic outcomes become almost incomprehensible. The range of phenotypic expressions in a given environment has been referred to as the 'reaction norm' [15]. The reaction norms arising from UV-radiation doses have yet to be worked out in models involving adaptive dynamics [16, 17].

In California, coniferous trees dating nearly 5000 years of age have survived at very high elevations where UV-B radiation exposure is high. Live cells are supported by their largely adaptive woody structures comprising dead cells (e.g., tracheids). We do not yet know how species at high elevations are programmed to avoid DNA damage or to express cell death under UV radiation. DNA polymerase 8 is required for DNA replication. Replication in plant and animal cells is dependent on the proliferating cell nuclear antigen (PCNA). PCNA activity was useful in distinguishing which conifer cells in a population express apoptosis leading to xylogenesis and/or tracheid formation [18].

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